Abstract

OBJECTIVE An emerging model of metabolic syndrome and type 2 diabetes is of adipose dysfunction with leukocyte recruitment into adipose
leading to chronic inflammation and insulin resistance (IR). This study sought to explore potential mechanisms of inflammatory-induced
IR in humans with a focus on adipose tissue.

CONCLUSIONS We demonstrate, for the first time in humans, that acute inflammation induces systemic IR following modulation of specific
adipose inflammatory and insulin signaling pathways. It also provides a rationale for focused mechanistic studies and a model
for human proof-of-concept trials of novel therapeutics targeting adipose inflammation in IR and related consequences in humans.

Adipose dysfunction, insulin resistance (IR), and type 2 diabetes are proinflammatory states. Indeed, gene manipulation of
Toll-like receptors (1,2), chemokines (3,4), and cytokines in experimental models has defined a role of innate and adaptive immunity in diet-induced adipose dysfunction
and IR. An emerging model is one of early adipose recruitment of T-cells and macrophages with adipocyte inflammation and resistance
to insulin-promoting metabolic syndrome, type 2 diabetes, and atherosclerosis.

Experimental data suggest that inflammation may attenuate adipocyte insulin signaling. Dietary and inflammatory activation
of the inhibitor of nuclear factor κB kinase β-subunit (IKKβ), c-Jun NH2-terminal kinase (JNK) (5), protein kinase C (PKC) (6), and janus tyrosine kinases (JAK)/signal transducers and activators of transcription (STAT) (7) have been implicated in IR in adipocytes and rodent experimental models (7). These inflammatory kinases may promote IR by downregulating components of the insulin signaling cascade, including the
insulin receptor and insulin receptor substrates (IRSs), and by inducing suppressors of cytokine signaling (SOCS), inhibitors
of insulin receptor signaling. Despite substantial data in animal models, however, little is known of the specific role and
mechanisms of inflammatory IR in humans. Defining whether adipose inflammation occurs in human IR and which specific pathways
are involved will provide greater insight into human pathophysiology and inform preventive and therapeutic strategies for
type 2 diabetes and its complications.

We and others utilize low-dose experimental endotoxemia to activate Toll-like receptor (TLR)-4 signaling in vivo as a model
of inflammation-induced metabolic disturbances in humans (8–10). Here, we define the effects of endotoxemia on insulin sensitivity and focus on adipose inflammation because of its emerging
relevance in dietary excess and adipose dysfunction in human IR. We demonstrate, for the first time in humans, that activation
of innate immunity in vivo induces IR following modulation of specific adipose inflammatory and insulin signaling pathways.

RESEARCH DESIGN AND METHODS

Healthy volunteers were recruited from the general population of the Delaware Valley (10). The protocol was approved by the institutional review board of the University of Pennsylvania, and subjects gave written
informed consent. Full details are described in the online appendix (available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0367/DC1). Briefly, criteria included healthy men or nonpregnant/lactating women, aged 18–40 years, with BMI of 18–30 kg/m2. Twenty subjects were recruited, equally divided by sex, to the University of Pennsylvania's Clinical Translational Research
Center for three visits: visit 1 for screening; visit 2, 2 weeks later, for frequently sampled intravenous glucose tolerance
(FSIGT) testing and dietary counseling; and visit 3, consisting of an overnight acclimatization phase, a 24-h saline control
phase, and a 24-h post-lipopolysaccharide (LPS) study phase (60-h total). LPS (U.S. standard reference endotoxin, lot no.
CC-RE-LOT-1 + 2; Clinical Center, Pharmacy Department, National Institutes of Health) was given intravenously as a 3 ng/kg
bolus at 0600 h on day 2. Blood samples (nine before and nine after LPS) and subcutaneous gluteal adipose aspiration-biopsy
samples (before and 4, 12, and 24 h after LPS) were collected (in n = 17 participants; 621.4 ± 253.1 mg average weight per sample).

Laboratory measures.

Two weeks prior to LPS and 24 h following LPS, the insulin sensitivity index (Si) was derived from an FSIGT test. We chose the 24-h post-LPS because we expected IR to be established by this time point based
on human and animal experimental and observational data (8,10–12) and because of practical considerations given the experimental design. We chose FSIGT as the method for determining insulin
sensitivity because the test also provides a measure of pancreatic β-cell function, the acute insulin response to glucose
(AIRg), and so allows assessment of endotoxemia effects on the β-cell. The FSIGT test was conducted using the insulin-modified
approach as previously described (13). Si was derived from Bergman's minimal model (14) using MINMOD Millennium software (15). AIRg was calculated as the incremental area under the curve for insulin from t = 0 to 10 min (12). Complementary estimates of IR and β-cell function, the homeostasis model assessment for IR (HOMA-IR) index [glucose (mmol/l)
× insulin (μU/ml)/22.5], and the HOMA for β-cell function (HOMA-B) index [insulin (μU/ml) × 20/glucose (mmol/l) − 3.5] were
calculated using fasting glucose and insulin values at 24 h and 5 min before and 24 h after LPS. Plasma biomarkers and lipoprotein
measurement was described previously (10) and is outlined in the online appendix.

Statistical analysis.

Data are reported as means ± SE for continuous variables and as proportions for categorical variables. In general, the effect
of endotoxemia on plasma biomarkers, metabolic measures, blood and adipose mRNAs, and adipose protein levels were tested by
mixed-effects modeling or repeated-measures ANOVA. For plasma data, we considered the time-matched difference (after minus
before for matched time points prior to and following LPS) in biomarker responses and used a mixed-effects model to consider
the effect due to LPS. A variety of models including linear, quadratic, and cubic polynomial were fit, and the best fit for
each variable was used for final analyses (e.g., quadratic models for TNF, IL-6, and resistin). A similar time-matched mixed-effect
modeling approach was applied to whole-blood mRNA data. Repeated-measures ANOVA was applied to FSIGT data (Si and AIRg), HOMA data (HOMA-IR and HOMA-B), and adipose mRNA data. When significant global differences were found in ANOVA, post hoc
paired t tests were used to compare time points. Analyses were performed using the freeware statistical package R (version 2.4.1; The R Foundation for Statistical Computing). Statistical significance was defined as a P value <0.05.

Systemic inflammatory and metabolic responses.

We have previously described plasma adipokine responses in this study sample (10). Briefly, endotoxemia induced a marked, rapid, and transient induction of plasma TNF and IL-6 (Table 2), followed by a robust increase in circulating resistin and a delayed but significant increase in leptin and leptin-soluble
leptin ratio in plasma. We now report significant increases in additional circulating inflammatory markers including MCP-1
and high-sensitivity C-reactive protein (hsCRP) (Fig. 1A) as well as plasma cortisol and free fatty acids, with a trend toward increased growth hormone (GH) (P = 0.08) levels (Fig. 1B). Overall, these findings confirm a transient inflammatory response during human endotoxemia with modulation of several inflammatory,
adipokine, and hormonal cascades that are known to impact insulin sensitivity.

IRS family members are key mediators of cellular insulin signaling. Prior to LPS, we detected mRNA for IRS-1 and IRS-2 but
not IRS-3 or IRS-4 in adipose with differential expression compared with whole blood (supplementary Table 2). Endotoxin reduced
IRS-1 (by 47%) mRNA in adipose. In whole blood, IRS-1 mRNA levels also fell (by 53%), but IRS-2 mRNA increased (2.3-fold increase)
(Table 3). In parallel, insulin receptor mRNA levels decreased significantly in blood but not in adipose, and there was no significant
change in GLUT4 mRNA in adipose or whole blood (Table 3). Western blotting of adipose proteins generally supported mRNA data with reduction in IRS-1 but no change in insulin receptor;
however, GLUT4 protein levels tended to fall (Fig. 5). Furthermore, enhanced serine phosphoryation of AKT, with no change in total AKT (Fig. 5), was observed consistent with previous findings of TNF-induced serine phosphorylation of AKT in adipocytes (17). Overall, endotoxemia induced specific SOCS proteins and modulated several components of the insulin signaling pathway in
adipose.

DISCUSSION

Inflammation, particularly in adipose tissue, has been implicated in diet- and obesity-related IR in experimental models.
Resistance to insulin also occurs acutely in human states of infection and sepsis. However, the specific mechanisms and the
potential for therapeutic targeting in humans are poorly understood. In this work, we found that endotoxemia induced systemic
IR but not pancreatic β-cell dysfunction in humans. Further, IR measured at 24 h post-LPS was preceded by specific modulation
of adipose inflammatory and insulin signaling pathways. This work defines specific targets for inflammatory modulation of
insulin signaling in humans and also provides a human model for proof-of-concept studies of novel therapeutics in IR and its
complications.

Epidemiological studies (18,19) suggest causal links between chronic inflammation, IR, and incident type 2 diabetes, while observational data demonstrate
that IR and overt type 2 diabetes may emerge during human infections and sepsis (11). Agwunobi et al. (8) were the first to show impaired insulin sensitivity 6–7 h following LPS administration utilizing euglycemic clamp studies.
Our study goes beyond the findings of Agwunobi et al. by demonstrating persistence of IR at 24 h after endotoxin in the absence
of any effect on pancreatic β-cell function while also identifying adipose tissue inflammatory responses and modulation of
specific adipose insulin signaling proteins that precede systemic IR. Interestingly, Agwunobi et al. also noted enhanced insulin
sensitivity 2 h after LPS as determined by a significant increase in the glucose infusion rate required during the clamp.
A recent elegant study (20) using isotope tracers with a euglycemic clamp showed that this acute and transient increase in insulin sensitivity at 1–2
h after LPS was due to increases in both hepatic and peripheral insulin sensitivity.

Experimental models support an important role for innate and adaptive immunity in diet- and obesity-induced IR (1,3–5,21). Deficiency of TLR-4, the innate antigen/LPS receptor, protects against diet-induced obesity and IR in rodents (2). TNF impairs insulin-mediated glucose disposal, and functional TNF deficiency in mice protects from obesity-induced IR (21). However, the relevance to human pathophysiology of individual signaling pathways implicated in rodent models remains unknown.
In fact, species heterogeneity in inflammatory modulation (22) and of insulin signaling has been documented (9,23). Thus, use of human models of inflammation can provide unique insight into clinically relevant mechanisms and therapeutic
targets for IR and type 2 diabetes.

Our study is the first to demonstrate loss of insulin sensitivity without any apparent effect on pancreatic β-cell function
during acute human inflammation. Because fasting-based HOMA-IR estimates have been shown to correlate best with measures of
hepatic insulin sensitivity and FSIGT Si with measures of peripheral insulin sensitivity (24), endotoxemia appears to trigger both hepatic and peripheral IR. Indeed, we note that Agwunobi et al. (8) published data with euglycemic clamps that demonstrate hepatic IR following LPS. Further, we describe several inflammatory
perturbations that may impact tissue and systemic insulin sensitivity, induction of inflammatory cytokines and chemokines,
modulation of adipokine signaling (10), activation of the hypothalamic-pituitary-adrenal axis (25), and altered flux of plasma free fatty acids (26). Indeed, the degree of evoked change in several inflammatory and metabolic markers, including free fatty acids, hsCRP, resistin,
and GH tended to precede and correlate with the degree of IR. Taken together, therefore, these data support a model of both
hepatic and peripheral IR during endotoxemia, with peripheral IR likely to be occurring at the level of skeletal muscle as
well as adipose tissue.

Recent experimental studies in rodents demonstrated that adipose recruitment of T-cell and macrophages in obesity promotes
adipocyte inflammation leading to local and systemic IR (27). We hypothesized that adipose inflammation would be a consequence of human endotoxemia that might contribute to local and
systemic IR. Endotoxemia induced a rapid and transient increase in adipose TNF and IL-6. There was also a marked induction
of adipose MCP-1, which is known to recruit chemokine CC motif receptor (CCR)-2–expressing monocytes, increase inflammatory-M1
adipose tissue macrophage (ATM), and promote IR (27). Recent studies of diet-induced obesity suggest that upregulation of T-cell chemokines in adipose and recruitment of inflammatory
TH1 cells precedes recruitment of monocytes and the development of systemic IR. Remarkably, we found that endotoxemia induced
CXCL10, a potent T-cell chemokine. The emergence of resistin mRNA in adipose suggests leukocyte recruitment because expression
of this adipokine is restricted to myeloid lineage in humans (23). In addition, we found increased mRNA levels of the macrophage marker EMR1-F4/80 (28) in adipose, further supporting that endotoxemia may promote adipose recruitment of macrophages. Overall, these data suggest
that endotoxemia induces human adipose inflammatory responses similar to those observed in models of diet- and obesity-related
IR (1–4,21,27).

Whether inflammation attenuates adipose insulin signaling in humans and which signaling pathways are involved has not been
defined. Several inflammatory adipokines such as TNF, IL-6, and resistin, as well as endotoxin itself, induce SOCS proteins
that inhibit insulin receptor signaling and target IRS proteins for ubiquitination and proteosomal degradation (29,30). The SOCS family, consisting of eight members, is recognized as a general negative feedback mechanism for receptor tyrosine
kinase signaling including the insulin receptor. Using the yeast two-hybrid system, SOCS-1, SOCS-3, and SOCS-6 have been shown
to bind to the insulin receptor (31), and cells from SOCS-1–deficient mice exhibit enhanced insulin sensitivity (30). Conversely, in obesity, SOCS-1 and SOCS-3 are increased in liver, muscle, and fat coincident with reduced tyrosine phosphorylation
of IRS proteins. We report for the first time the pattern of SOCS family mRNA expression in human adipose and a marked and
selective induction of adipose SOCS proteins during endotoxemia; SOCS-1 and SOCS-3 were increased with no effect of SOCS-2
and SOCS-6. Our findings suggest that induction of SOCS-3 in adipose may be an important molecular mechanism of IR in human
inflammatory states (31).

The in vivo effect of inflammation on insulin receptor signaling in human adipose is unknown. The insulin receptor, a transmembrane
dimeric protein with intrinsic kinase activity, recruits IRS proteins upon insulin binding. Tyrosine phosphorylation of IRS
proteins activates phosphatidylinositol-3-kinase, leading to AKT phosphorylation and GLUT4 mobilization (32). Inflammatory kinases including IKKβ (5,33), JNK (5), PKCs (6), and JAK-STATs attenuate insulin signaling in adipocytes and in rodent models. These kinases induce serine phosphorylation
of IRS-1, which inhibits IRS-1 tyrosine phosphorylation during insulin signaling (32). We found tissue-specific IRS expression and downregulation of adipose IRS-1 protein coincident with reduced IRS-1 mRNA.
Our data also suggest species heterogeneity in the pattern of adipose IRS expression with more abundant IRS-2 in human adipose
compared with that reported in rodents (32). The effect of endotoxemia on IRS-1 protein levels is one of several mechanisms by which endotoxemia may impair insulin
signaling in human adipose. Remarkably, changes in several mRNAs in whole blood paralleled that in adipose (e.g., IL-6, MCP1,
SOCS-1, and SOCS-3). However, inflammation appears to modulate specific insulin signaling–related proteins (insulin receptor,
SOCS-2 and SOCS-6, and IRS-2) in a tissue-specific manner. This should prompt caution in extrapolating tissue-specific effects
from global characterization of whole-blood mRNAs.

Overall, endotoxemia induces IR in humans following modulation of adipose tissue inflammatory and insulin signal pathways
in vivo. While adipose dysfunction in genetically and environmentally susceptible patients may increase inflammation, experimental
endotoxemia may also induce adipose inflammation and subsequent adipocyte dysfunction, which then leads to IR. However, our
study has several limitations. While we have not definitively proven that adipose inflammation is causal in systemic IR during
endotoxemia, our work provides proof of principle that inflammation-induced systemic IR emerges after inflammatory modulation
of adipose insulin signaling in humans. We emphasize the need for specific study of the chronic low-grade human inflammation
observed in obesity, metabolic syndrome, and type 2 diabetes. Our approach to study insulin sensitivity using the FSIGT-derived
Si at 24 h post-LPS is limited in that we cannot differentiate various contributions of changes in hepatic and peripheral insulin
sensitivity versus the total body change and cannot define the kinetics of the development and resolution of IR. However,
we utilized the FSIGT Si as a sensitive measure of peripheral IR and combined this with the HOMA-IR that correlates best with measures of hepatic
insulin sensitivity (24). Furthermore, FSIGT enabled examination of whether pancreatic β-cell function changes during inflammation-induced IR, while
the hyperinsulinemic-eugylemic clamp does not. We acknowledge that inflammatory effects are likely to occur in liver and in
skeletal muscle during endotoxemia and that these could impact systemic IR. Indeed, our HOMA-IR data and our FSIGT data support
both hepatic and peripheral IR consistent with published euglycemic clamp studies. Detailed examination of changes in skeletal
muscle and greater study of adipose tissue inflammation and function in relationship to the kinetics of IR is warranted in
future studies. Despite these limitations, our study provides the first tissue level data on evoked inflammatory pathways
in human IR.

Finally, endotoxemia may not represent accurately the pathophysiology of chronic inflammatory, insulin-resistant disease states.
Several lines of evidence, however, support its relevance to the pathophysiology of IR in humans. First, an inflammatory IR
and metabolic dyslipidemia emerges clinically during acute sepsis (11) and chronic infections (34). Second, we and others have shown that cytokine/adipokine (10,23,35), acute-phase reactant responses, and lipoprotein changes (36,37) observed acutely during experimental endotoxemia resemble those chronically observed in the metabolic syndrome. Third, gene
manipulation and drug targeting of the TLR-4 (2,38) and nuclear factor κB (5,39) have provided proof of concept that modulation of innate immune signaling attenuates IR and type 2 diabetes in dietary and
obesity models. Last, and directly relevant to the effect on adipose, we recently demonstrated that endotoxemia induces gene
expression responses in subcutaneous adipose (40) that are remarkably similar to the changes observed in visceral adipose in insulin-resistant states (41–43).

Conclusion.

Human endotoxemia induces systemic IR but not pancreatic β-cell dysfunction. Remarkably, evoked adipose inflammation and modulation
of adipose insulin signal pathways, similar to some of those described in rodent models of diet-induced obesity and IR, precede
the emergence of systemic IR in humans. Our findings suggest specific targets in humans that warrant further mechanistic focus.
For example, induction of specific SOCS proteins and downregulation of IRS-1 are likely to play roles in the inflammatory
induction of adipose and systemic IR in humans. This work also provides a human experimental model for studies of novel therapeutics
targeting systemic and adipose inflammation in IR and its metabolic consequences.

Acknowledgments

This work was supported by a Clinical and Translational Science Award (UL1RR024134) from the National Center for Research
Resources (NCRR) and a Diabetes and Endocrine Research Center (P20-DK019525) award, both to the University of Pennsylvania.
M.P.R. is also supported by RO1 HL-073278 and P50 HL-083799-SCCOR from the National Institutes of Health. N.N.M. is a recipient
of the ACC Young Investigator Award for the metabolic syndrome.

No potential conflicts of interest relevant to this article were reported.

Footnotes

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